This invention relates, in part, to newly identified polynucleotides and polypeptides; variants and derivatives of the polynucleotides and polypeptides; processes for making the polynucleotides and the polypeptides, and their variants and derivatives; agonists and antagonists of the polypeptides; and uses of the polynucleotides, polypeptides, variants, derivatives, agonists and antagonists. In particular, in these and in other regards, the invention relates to polynucleotides and polypeptides of human endometrial specific steroid-binding factor I, II and III, sometimes hereinafter referred to as xe2x80x9chESF I, II and IIIxe2x80x9d.
The regulation of cells and tissues is controlled by autocrine and paracrine factors, such as systemic hormones and factors that modulate or mediate the action of hormones.
Many peptides, expressed locally, can influence certain biological activity in the mammalian system and are very important in the regulation of cells of the epithelium. These factors largely have not been identified or characterized, particularly not in humans.
A few factors that play a role in the regulation of functions of the lung and uterus, both adult and fetal, have been identified in non-human organisms. One such factor is mammalian CC10, i.e., human, rat and rabbit CC10. (Wolf, M. et al., Human Molecular Genetics, 1(6):371-378 (1992)). Clara Cell 10 kDa secretory protein (CC10) which is also called Clara Cell 17 kDa protein, is a homodimer consisting of 8.5 kDa monomers that are joined by two disulfide bonds (Umland, T. C. et al., J. Mol. Biol., 224:441-448 (1992)). It is the predominant secreted protein of lung Clara cells which are the lining of the bronchiolar epithelium (Singh, G. and Katyal, S. L., J. Histochem. Cytochem., 32:49-54 (1984)). The physiological role of the protein is not yet completely understood. It has been reported that CC10 specifically binds methylsulfonyl-polychlorated biphenyls (PCBs) (Nordlund, Moler, L. et al., J. Biol. Chem., 265:12690-12693 (1990)) and inhibits phospholipase A2 (Singh, G. et al., Biochem. Biophys. Acta, 1039:348-355 (1990)). In the last few years the sequences of rat (Katyal, S. L. et al., Prog. Respir. Res., 25:29-35 (1990); and Hagen, G. et al., Nucleic Acids Res., 18:2939-2946 (1990)), and human (Singh, G. et al., Biochem. Biophys. Acta, 950:329-337 (1988) CC10 cDNAs have been reported. cDNAs, and the derived amino acid sequences, show striking homologies to rat uteroglobin (Singh, G. et al., Biochem. Biophys. Acta, 1039:348-355 (1990); and Hagen, G. et al., Nucleic Acids Res., 18:2939-2946 (1990)).
Like CC10, rat uteroglobin is a covalently bound homodimer whose three dimensional structure is well known (Morize, I. et al., J. Mol. Biol., 194:725-739 (1987). Uteroglobin expression in rabbits has been originally reported in the uterus during the preimplantation phase (Beier, H. M., Biochem. Biophys. Acta, 160:289-290 (1968)). More recently, the protein was also detected in oviduct (Kirchner, C., Cell Tissue Res., 170:490-492 (1976)), male genital organs (Beier, H. M. et al., Cell Tissue Res., 165:1-11 (1975)), esophagus (Noske, I. G. and Feigelson, M., Biol. Reprod., 15:704-713 (1976)) and lung (Noske, supra; and Torkkeli, T. et al., Biochem. Biophys. Acta, 544:578-592 (1978)).
In vitro, several distinct properties of uteroglobin have been described. Soon after its discovery it could be shown that the steroid hormone progesterone is specifically bound by the protein (Beato, M. and Baier, R., Biochem. Biophys. Acta, 392:346-356 (1975); and Beato, M. et al., J. Steriod Biochem., 8:725-730 (1977)). Therefore, rabbit uteroglobin was believed to be a potential carrier or scavenger of progesterone that regulates the progesterone concentration in the endometrium (Atger, M. et al., J. Steroid Biochem., 13:1157-1162 (1980)). It has also been shown to specifically bind certain methylsulfonyl metabolites of polychlorinated biphenyls with even higher affinity than progesterone (Gillner, M. et al., J. Steroid Biochem., 31:27-33 (1988)). Furthermore, uteroglobin has been found to inhibit phospholipase A2. The relationships of all these properties and their physiological significance is still not understood and remains largely a matter of speculation.
The rat CC10 mRNA is expressed like rat uteroglobin not only in lung but also in the esophagus as well in uteri of estrogen and progesterone treated female rats (Hagen, G. 1990 supra) suggesting that rat CC10 is the rat counterpart of rat uteroglobin (see in general Wolf, M. et al., Human Molecular Genetics, 1(6):371-378 (1992)).
Human CC10 expression is abundant in non-neoplastic human lung, and it is detectable in tumors in corresponding cell lines at markedly lower levels (Broers, J. L. V. et al., Lab. Invest., 66:337-346 (1992); Linnoila, R. I. et al., Amer. J. Clin. Pathol., 90:1-12 (1988)). CC10 levels were also significantly lower in 6 serum and bronchoalveolar lavage specimens obtained from smokers and lung cancer patients compared with specimens from healthy non-smokers (Bernard, A. et al., Europ. Resp. J., 5:1231-1238 (1992)). These findings suggest the expression of CC10 mRNA becomes altered in distinct lung compartments and may implicate a role for CC10 in the development of pulmonary carcinomas (Jensen, S. M. et al., Int. J. Cancer, 58:629-637 (1994).
Some of the biological properties of UG, such as masking the antigenicity of blastomers (Mukherjee, A. B., et al., Med. Hypotheses, 6:1043-1055 (1980)) and epididymal spermatozoa (Mukherjee, D. C., et al., Science (Wash. D.C.), 219:989-991 (1983)), inhibition of monocyte and neutrophil chemotaxis and phagocytosis in vitro (Schiffman, E. V., et al., Agents Actions Suppl., 12:106-120 (1983)), and inhibition of ADP- and thrombin-induced (but not of arachidonic acid-induced) platelet aggregation (Manjunath, R., et al., Biochem. Pharmacol., 36:741-746 (1987)), may be due, at least in part, to the potent inhibitory effect of this protein on PLA2 activity (Levin, S. W., et al., Life Sci., 38:1813-1819 (1986)). A nonapeptide derived from the amino acid sequence of xcex1-helix-3 of UG monomer (residues 39-47) possesses all the biological properties of the intact protein and has been identified as an active site of UG responsible for its PLA2-inhibitory and antiinflammatory activities (Miele, L., et al., Nature (Lond.), 335:726-730 (1988)).
It has been indicated that cclOkD-specific transcripts are present in several nonrespiratory human organs and tissues. By using an antibody to rabbit UG, a UG-like immunoreactivity in human endometrium (Kikukawa, T., et al., J. Clin. Endocrinol. Metab., 67:315-321 (1988)), prostate (Manyak, M. J., et al., J. Urol., 140:176-182 (1988)), and respiratory tract (Dhanireddy, R., et al., Biochem. Biophys. Res. Commun., 152:1447-1454 (1988)), has been described.
Recently, the cDNA (Singh, G., et al., Biochem. Biophys. Acta, 950:329-337 (1988)) and the 5xe2x80x2 regions (Wolf, M., et al., Human Mol. Genet., 1:371-378 (1992)) of the gene encoding human uteroglobin (hUG), a counterpart of rabbit UG (rUG), has been characterized. Human UG or Clara cell 10-kD protein has 61.5% amino acid sequence identity with rUG (Singh, G., et al., Biochem. Biophys. Acta, 950:329-337 (1988)), 54.2% similarity with rat UG (Singh, G., et al., Biochem. Biophys. Acta, 1039:348-355 (1990)), and 52.8% with mouse UG (Singh, G., et al., Exp. Lung Res., 19:67-75 (1993)). Although this protein was originally discovered in the alveolar Clara cells (Singh, G., et al., J. Histochem., 36:73-80 (1988)) it is detectable in many extrapulmonary tissues similar to the ones in which rUG is expressed (Peri, A., et al., DNA Cell Biol., 5:495-503 (1994)) and this expression is induced by progesterone. It appear that some of the biological properties of hUG are virtually identical to rUG (Mantile, G., et al., J. Biol. Chem., 27:20343-20351 (1993)).
It has been reported that UG in the rabbit uterine fluid is first detectable on day 3 of pregnancy, and peak level is reached on day 5 (for a review see Miele, L., et al., Endocr. Rev., 8:474-490 (1987)). UG, by inhibiting PLA2 activity, may down-regulate the production of proinflammatory lipid mediators, which promote contraction and motility of the uterine smooth muscle. Therefore, it is suggested that UG facilitates the maintenance of myometrial quiescence during gestation.
There is a clear need in the art to further isolate and characterize proteins which are homologues of mammalian Clara cell 10 kDa secretory protein and rat prostatic steroid-binding protein. The genes and gene products of the present invention display homology to the rat prostatic steroid-binding protein and Clara cell 10 kDa secretory protein.
Toward these ends, and others, it is an object of the present invention to provide polypeptides, inter alia, that have been identified as novel hESF I, II and III by homology between the amino acid sequence set out in FIGS. 1, 2 and 3 (SEQ ID NO:2, 4 and 6) and known amino acid sequences of other proteins such as rat prostatic steroid-binding protein.
It is a further object of the invention, moreover, to provide polynucleotides that encode hESF I, II and III, particularly polynucleotides that encode the polypeptides herein designated hESF I, II and III.
In a particularly preferred embodiment of this aspect of the invention the polynucleotides comprise the regions encoding human hESF I, II and III in the sequence set out in FIGS. 1, 2 and 3 (SEQ ID NO:2, 4 and 6).
In accordance with this aspect of the present invention there is provided isolated nucleic acid molecules encoding mature polypeptides expressed by the human cDNA contained in ATCC Deposit No. 97401 (ESF I), 97402 (ESF II) and 97403 (ESF III).
In accordance with this aspect of the invention there are provided isolated nucleic acid molecules encoding human hESF I, II and III, including mRNAs, cDNAs, genomic DNAs and, in further embodiments of this aspect of the invention, biologically, diagnostically, clinically or therapeutically useful variants, analogs or derivatives thereof, or fragments thereof, including fragments of the variants, analogs and derivatives.
Among the particularly preferred embodiments of this aspect of the invention are naturally occurring allelic variants of human hESF I, II and III.
It also is an object of the invention to provide hESF I, II and III polypeptides, particularly human hESF I, II and III polypeptides, that treat and/or prevent inflammation, asthma, rhinitis, cystic fibrosis, airway disease, neoplasia, atopy, inhibit phospholipase A2 activity, bind polychlorinated biphenyls, reduce foreign protein antigenicity, inhibit monocyte and neutrophil chemotaxis and phagocytosis, inhibit platelet aggregation, regulate eicosanoid levels in the human uterus and control the growth of endometrial cells.
In accordance with this aspect of the invention there are provided novel polypeptides of human origin referred to herein as hESF I, II and III as well as biologically, diagnostically or therapeutically useful fragments, variants and derivatives thereof, variants and derivatives of the fragments, and analogs of the foregoing.
Among the particularly preferred embodiments of this aspect of the invention are variants of human hESF I, II and III encoded by naturally occurring alleles of the human hESF I, II and III genes.
It is another object of the invention to provide a process for producing the aforementioned polypeptides, polypeptide fragments, variants and derivatives, fragments of the variants and derivatives, and analogs of the foregoing. In a preferred embodiment of this aspect of the invention there are provided methods for producing the aforementioned hESF I, II and III polypeptides comprising culturing host cells having expressibly incorporated therein an exogenously-derived human hESF I, II or III encoding polynucleotide under conditions for expression of human hESF I, II and III in the host and then recovering the expressed polypeptides.
In accordance with another object of the invention there are provided products, compositions, processes and methods that utilize the aforementioned polypeptides and polynucleotides for research, biological, clinical and therapeutic purposes, inter alia.
In accordance with certain preferred embodiments of this aspect of the invention, there are provided products, compositions and methods, inter alia, for, among other things: assessing hESF I, II and III expression in cells by determining hESF I, II and III polypeptides or hESF I, II and III-encoding mRNA; expressing hESF I, II and III in vitro, ex vivo or in vivo by exposing cells to hESF I, II and III polypeptides or polynucleotides as disclosed herein; assaying genetic variation and aberrations, such as defects, in hESF I, II and III genes; and administering a hESF I, II and III polypeptide or polynucleotide to an organism to augment hESF I, II and III function or remediate hESF I, II and III dysfunction.
In accordance with certain preferred embodiments of this and other aspects of the invention there are provided probes that hybridize to human hESF I, II and III sequences.
In certain additional preferred embodiments of this aspect of the invention there are provided antibodies against hESF I, II and III polypeptides. In certain particularly preferred embodiments in this regard, the antibodies are highly selective for human hESF I, II and III.
In accordance with another aspect of the present invention, there are provided hESF I, II and III agonists. Among preferred agonists are molecules that mimic hESF I, II and III, that bind to hESF I, II and III-binding molecules or receptor molecules, and that elicit or augment hESF I, II and III-induced responses. Also among preferred agonists are molecules that interact with hESF I, II and III polypeptides, or with other modulators of hESF I, II and III activities, and thereby potentiate or augment an effect(s) of hESF I, II and III.
In accordance with yet another aspect of the present invention, there are provided hESF I, II and III antagonists. Among preferred antagonists are those which mimic hESF I, II and III so as to bind to hESF I, II and III receptors or binding molecules but not elicit a hESF I, II and III-induced response or more than one hESF I, II and III-induced response or which prevent expression of hESF I, II and III. Also among preferred antagonists are molecules that bind to or interact with hESF I, II and III so as to inhibit an effect(s) of hESF I, II and III.
The agonists and antagonists may be used to mimic, augment or inhibit the action of hESF I, II and III polypeptides. They may be used, for instance, to treat and/or prevent an inherited susceptibility to asthma.
In a further aspect of the invention there are provided compositions comprising a hESF I, II or III polynucleotide or a hESF I, II or III polypeptide for administration to cells in vitro, to cells ex vivo and to cells in vivo, or to a multicellular organism. In certain particularly preferred embodiments of this aspect of the invention, the compositions comprise a hESF I, II or III polynucleotide for expression of a hESF I, II or III polypeptide in a host organism for treatment of disease. Particularly preferred in this regard is expression in a human patient for treatment of a dysfunction associated with aberrant endogenous activity.
Other objects, features, advantages and aspects of the present invention will become apparent to those of skill from the following description. It should be understood, however, that the following description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following description and from reading the other parts of the present disclosure.